Cryptococcus neoformans is an opportunistic pathogen that is responsible for life-threatening disease in patients with AIDS or other conditions of immunocompromise; extrapulmonary cryptococcal infection is an AIDS-defining illness. The long-term goal of our studies is to understand the pathogenesis of cryptococcosis. This application focuses on a fundamental part of this process, the interactions of this facultative intracellular pathogen with the phagocytic cells of its mammalian host. These interactions resolve with varying levels of damage to the host and yeast, but the mechanisms involved are poorly understood, limiting our ability to influence resolution in favor of the host. We propose to combine forward genetics, high-throughput screening assays, and focused follow-up studies to determine the cryptococcal factors that are required for this pathogen to successfully adhere to and enter host cells. Aim I is to identify mutants in these processes, using insertional mutagenesis and automated screening. Aim II is to determine the mechanisms by which selected gene products influence pathogen adherence and entry interactions. These studies will employ assays of cell adhesion, entry, and intracellular survival and growth; molecular techniques for sequence recovery, analysis, and gene deletion; and phenotypic assessment including morphological studies, biochemical analysis, and virulence assays. Results will be assessed using rigorous statistical methods and appropriate controls, and will be interpreted in the context of our expertise in the fundamental biology of Cryptococcus. The proposed studies will elucidate a crucial aspect of pathogenesis that determines the survival and dissemination of the organism and the host's ability to limit disease. This work will additionally open new areas for investigation of an important pathogen and develop experiment tools that can be applied to other areas of fungal biology.